This project is designed to develop a new approach to cancer treatment through the study of growth, survival, and metastasis regulatory signal transduction events that identify molecular targets for anticancer drug development. Our work is divided into basic research and translational research through the Preclinical Development Research Core, a translational drug development facility that we have established. Our work is currently focused on (1) histone deacetylase as a target for anticancer drug development, and (2) the molecular mechanisms of hematopoietic cell regulation by beta-catenin and the identification of beta-catenin as a target in hematologic malignancies (3) development of novel pharmacodynamic assays, including assays for antiangiogenic therapy. (1) Our basic research on signal transduction pathways that can inhibit the growth of hormone-refractory prostate cancer cells led us to the identification of histone deacetylase as a critical target in this neoplasm. During this fiscal year we have finished development of a novel pharmacodynamic assay for assessment of HDAC inhibitor activity in vivo. The NCI has applied for a patent for our work, which is uniquely capable of analyzing HDAC inhibitor activity in as little blood as in a finger-stick, and can look at combination therapy pharmacodynamic responses by examining 7 parameters simultaneously. We have used this technology in 3 clinical trials, two of which have been written for publication. We have established a collaboration with Drs. Jay Bradner and Stuart Schreiber of the Broad Institute to use our technology to develop new HDAC inhibitors. (2) While studying the anticancer action of lovastatin, a drug that was brought to Phase I clinical trial at the NCI as a direct translation of our research, we found that a critical determinant of sensitivity to the proapoptotic activity of lovastatin was the integrity of beta-catenin protein. This led us to examine the role of beta-catenin in apoptosis. We used hematologic malignancies as our model and found that beta-catenin plays an unexpectedly vital role in these cells. Our data demonstrated that beta-catenin regulates leukemia cell survival, proliferation, and adhesive properties. These data identified beta-catenin as a novel target for anticancer drug development in hematologic malignancies. We had published previously that beta-catenin promotes proliferation of the adult T-cell leukemia (ATL) cell line HUT102. We have established a collaboration with John Janik and John Morris of the Metabolism Branch, NCI to study their ATL patients and investigate the mechanism of beta-catenin signaling in ATL. The acute clinical subtype of adult T-cell leukemia/lymphoma have a very poor prognosis, despite the fact that it has been known for decades that the etiologic agent of ATL is the HTLV-1 virus, and that HTLV-1-encoded Tax plays a key role in HTLV-1-induced malignant transformation. Although Tax plays a critical role in the initial transformation process, Tax expression is frequently undetectable in the acute form of ATL. Thus, targeting of Tax would not appear to present a viable strategy in the most advanced and rapidly progressive form of ATL. We have discovered that (1) primary acute ATL cells express beta-catenin, (2) beta-catenin expression occurs in the absence of the Tax oncoprotein, (3) beta-catenin protein localizes to the cell nucleus in Tax-negative ATL cells, and (4) transcriptional analysis of primary ATL patient samples by our collaborator John Brady using Affymetrix arrays demonstrates high levels of expression of the beta-catenin transcriptional partner TCF4 and the beta-catenin/TCF4 target gene survivin. Recently survivin has been shown to be the most negative prognostic factor in ATL. We have succeeded in transfecting primary ATL cells, and have used this technique to transfect wild-type beta-catenin and a panel of constructs that block nuclear beta-catenin signaling as well as control siRNA and beta-catenin siRNA. These experiments demonstrated that in primary ATL cells survivin and the potent antiapoptotic gene Bfl-1 are under the transcriptional control of beta-catenin. Analysis of the pathways leading to beta-catenin overexpression and activation in primary ATL cells demonstrated a complex pattern of deregulatory events that stabilize beta-catenin and upregulate beta-catenin nuclear localization including Akt phosphorylation and CD45 silencing. Recently it has been demonstrated that NSAIDs such as celecoxib significantly down-regulate nuclear beta-catenin levels and block nuclear beta-catenin signaling. We screened a panel of NSAIDs against primary ATL cells and HTLV-1-infected cell lines and found that celecoxib had the most-favorable ratio of potency to toxicity, inhibited beta-catenin nuclear signaling and induced cell death. Together these data identify nuclear beta-catenin as a novel therapeutic target in advanced, Tax-independent ATLL. To pursue further our hypothesis that beta-catenin signaling is deregulated in hematologic malignancies, and that each malignancy is associated with a characteristic mechanism of deregulation, in collaboration with Tomohiro Kajiguchi of the Urologic Oncology Branch we have studied beta-catenin in two additional forms of leukemia, mast cell leukemia and FLT3 AML. We found that beta-catenin is a substrate for the tyrosine kinase c-kit, which is deregulated in mast cell leukemia. These experiments demonstrated that c-kit upregulates Wnt signaling in human mast cell leukemia, and that beta-catenin is a novel target for the treatment of mastocytosis and mast cell leukemia. This work is in press in Leukemia Research. FLT3 mutation or overexpression is the most common mutation in AML. We showed that FLT3 regulates beta-catenin tyrosine phosphorylation, nuclear localization, and target gene expression in FLT3-positive AML cells. This work was published in September 2007 in Leukemia. (3) The Preclinical Development Research Core has been working with intramural investigators on a range of phase I and phase II clinical trials. I am an associate investigator on 6 clinical trials. For each of these trials we work with the PI to develop novel pharmacodynamic endpoints, including analysis of circulating endothelial progenitor cells and mature endothelial cells. This year we have analyzed over 120 patients for these parameters. Our mulitparameter flow pharmacodynamic assay is in international phase patent filing and has been selected by intramural scientists for presentation at the NCI Technology Showcase September 25th, 2007. The flow assay was an integral part of the phase I trial of the HDAC inhibitor MS-275 published in Blood in April 2007, and in a second clinical trial of MS-275 in solid tumor patients, currently in press in Clinical Cancer Research. We have developed a new pharmacodynamic method, a cell-based tissue microarray for assessment of anticancer drug activity in vivo. The manuscript describing this technique is in press in Drug Development Research. We demonstrate the utility of this technique for analysis of protein hyperacetylation in response to treatment with the histone deacetylase inhibitor SNDX-275 in PBMC treated in vitro and in PBMC and bone marrow aspirates from patients on SNDX-275 phase I clinical trials. We demonstrate that the cell microarray can be used to measure drug response in a high-throughput manner, allowing analysis of an entire trial on one or two glass slides. The cell microarray technique brings the advantages of the tissue microarray platform to the pharmacodynamic assessment of single cells, such as those isolated from bone marrow aspirates, fine needle aspirates or malignant effusions, and to analysis of PBMC, the most commonly studied surrogate in oncology trials